Hematopoiesis ARTICLE Megakaryocyte volume modulates bone marrow niche properties and cell migration Ferrata Storti Foundation dynamics Maximilian G. Gorelashvili,1* Oğuzhan Angay,2* Katherina Hemmen,2 Vanessa Klaus,1 David Stegner1,2 and Katrin G. Heinze2 1Institute of Experimental Biomedicine, University Hospital Würzburg and 2Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany Haematologica 2020 *MGG and OA contributed equally to this work Volume 105(4):895-904 ABSTRACT ll hematopoietic cells that develop in the bone marrow must cross the endothelial barrier to enter t he blood circulation. Blood Aplatelets, however, are released by bigger protrusions of huge pro- genitor cells, named megakaryocytes, and enter the blood stream as so- called proplatelets before fragmenting into mature platelets. Recently, a second function of megakaryocytes has been identified, as they modu- late the quiescence of hematopoietic stem cells, mostly via different sol- uble factors. We know from light sheet fluorescence microscopy images that megakaryocytes are distributed throughout the bone marrow facing a dense vascular network. Here, we used such three-dimensional images to provide a realistic simulation template reflecting thein vivo cell-vessel distributions resulting in reliable whole-bone analysis in silico . Combining this approach with an automated image analysis pipeline, we found that megakaryocytes influence migration of neutrophils and Correspondence: hematopoietic stem cells, and thus act as biomechanical restrainers mod- KATRIN HEINZE ulating cell mobility and extravasation. Indeed, as a consequence of [email protected] increased megakaryocyte volumes in platelet-depleted mice neutrophil mobility was reduced in these animals. DAVID STEGNER [email protected] Received: July 15, 2018. Introduction Accepted: June 25, 2019. Pre-published: June 27, 2019. Hematopoiesis is the process of forming blood cells, which occurs predominant- ly in the bone marrow (BM). During this process, hematopoietic stem cells (HSC) undergo lineage restriction and differentiate into restricted hematopoietic progeni- doi:10.3324/haematol.2018.202010 tors, which then give rise to blood cells by proliferation and further differentiation. During the last decade, the identification of new HSC markers, development of Check the online version for the most updated genetically modified mouse strains and microscopy techniques enabled the identi- information on this article, online supplements, fication and intensive investigation of the perivascular niche as the major site of and information on authorship & disclosures: www.haematologica.org/content/105/4/895 HSC localization and activity, where 80% of HSC were found to be associated with sinusoids, 10% with arterioles and a further 10% with transition zone ves- sels.1-4 The perivascular niche has been determined to be a microenvironment con- ©2020 Ferrata Storti Foundation taining different cell types and signaling molecules, together regulating HSC main- tenance, quiescence, proliferation, differentiation and migration.5-8 Material published in Haematologica is covered by copyright. Blood cells differentiated from HSC leave the BM by migrating towards the sinu- All rights are reserved to the Ferrata Storti Foundation. Use of 9 published material is allowed under the following terms and soids to enter the circulation. In general, many of these migratory processes are conditions: chemotactic, i.e. driven by chemoattractants.9 One of the most prominent cell types https://creativecommons.org/licenses/by-nc/4.0/legalcode. of the BM are neutrophils, which are the most abundant subpopulation of leuko- Copies of published material are allowed for personal or inter- cytes; neutrophils have a short lifespan (6-12 h) in the circulation, after which they nal use. Sharing published material for non-commercial pur- 10 poses is subject to the following conditions: quickly migrate to tissues where they perform their functions. Neutrophil home- https://creativecommons.org/licenses/by-nc/4.0/legalcode, ostasis is orchestrated through a balance of neutrophil production and release from sect. 3. Reproducing and sharing published material for com- BM into the blood and migration back into the BM for elimination.11 The neutrophil mercial purposes is not allowed without permission in writing reserve within the BM is estimated to be 6×1011 in humans and 12×107 in mice.10,12 from the publisher. Megakaryocytes are derived from multipotent HSC and belong to the myeloid cell lineage.13 Megakaryocytes mainly reside in the BM, mostly in close proximity haematologica | 2020; 105(4) 895 M.G. Gorelashvili et al. to the vasculature,14,15 and have a diameter of up to 50 µm such simplified artificial templates for megakaryocytes in mice and 50-100 µm in humans.13 Mature megakary- and the vasculature can bias simulations and lead to mis- ocytes produce platelets and release them into the blood interpretations as we show in this study. Here, we demon- circulation in order to maintain constant platelet counts. In strate that using the segmented cell and vessel objects of addition, they actively regulate HSC proliferation in both true 3D images can overcome those limitations, providing positive and negative manners.16-19 Recent studies revealed a simulation framework that has the prerequisites to that most HSC are in close proximity to sinusoidal blood reflect the physiological situation optimally. vessels.1,4,20 Likewise, more than 70% of megakaryocytes were found to be in contact with the BM vasculature.15 Besides this indirect correlation, at least a subset of Methods megakaryocytes was found to be in close proximity to HSC.16,18,19 Moreover, megakaryocytes were shown to More methodological details are present in the Online influence HSC quiescence via different cytokines, such as Supplementary Material. 16 17 CXCL4, transforming growth factor β1 (TGFβ1) and thrombopoietin.18,21 However, very recently it was shown Mice that liver-derived, and not megakaryocyte-derived, throm- All animal experiments were approved by the district govern- bopoietin is required for HSC maintenance in the BM.22 ment of Lower Frankonia (Bezirksregierung Unterfranken). We Nevertheless, megakaryocyte activity in the intact used 8- to 12-week old C57BL/6JRj (Janvier Labs) or Lyz2GFP mice.34 medullary space and its interplay with other BM cells has Thrombocytopenia was induced by intravenous injection of rat 35 gained great attention in the last decade; numerous in vitro anti-GPIbα (CD42b; 2.0 mg/g body weight Emfret Analytics, investigations, based on two-dimensional (2D) cryosec- Eibelstadt, Germany;). tions, and in vivo (two-photon) imaging studies have been reported.23 Intrinsic limitations of these methods such as Two-photon intravital imaging loss of volume information, cutting artifacts or small field Lyz2GFP mice34 were anesthetized by intraperitoneal injection of of view impair scientific models15 and treatment of medetomidine 0.5 mg/g, midazolam 5 mg/g and fentanyl 0.05 patients.24 Recently, whole bone optical clearing and imag- mg/g body weight. A 1-cm midline incision was made to expose ing have been established to overcome these limitations. the frontoparietal skull, while carefully avoiding damage to the Despite the significant advances in imaging technology, bone tissue. The mouse was placed on a custom-designed metal tools for correct quantitative analysis of the geometry and stand equipped with a stereotactic holder to immobilize the head. localization of megakaryocytes, HSC and other BM com- BM vasculature was visualized by injection of bovine serum albu- ponents are still in their infancy. As image segmentation is min-Alexa546 (8 mg/g body weight) and anti-CD105 Alexa546 (0.6 a complex and error-prone method, exact definition of the mg/g body weight). Neutrophils were visualized by the endoge- image-processing pipeline is of great importance. The nously expressed green fluorescent protein. Stacks were acquired recently developed machine learning toolkits25,26 are pow- at a frame rate of 1/min on an upright two-photon fluorescence erful complements to the portfolio, and allow for more microscope (TCS SP8 MP, Leica Microsystems, Wetzlar, comprehensive data analysis, and access to previously Germany) equipped with a 25x water objective with a numerical masked information. Successful segmented objects aperture of 1.0. A tunable broad-band Ti:Sa laser (Chameleon, derived from complex microscopy data can be used for in Coherent, Dieburg, Germany) was used at 780 nm to capture silico analysis of cell distributions within the BM architec- green fluorescent protein and Alexa546 fluorescence. For each ture as recently demonstrated.15,16,20 mouse, three time series of z-stacks were recorded (20 min each, 3 Modular toolkits in particular have been proven to be 1 z-stack/min) with a voxel size of 0.87 x 0.87 x 1 mm . Details on powerful, not only for image analysis, but also for struc- image analysis are provided in the Online Supplementary Material. ture reconstruction as well as simulations of growth and organization.27,28 Unfortunately, these tools are not yet uni- Light sheet fluorescence microscopy image processing versally applicable. Here, we developed and compared dif- and segmentation ferent image processing pipelines and simulation scenarios Image stacks were processed, visualized, and analyzed using for precise identification of megakaryocytes in three- FIJI,36 Ilastik 1.2.25